Effects Of N-Glycosylation On Aspergillus Niger Phytase Characterization

Glycosylation was regarded as one of the most important posttranslational modifications in newly synthesized proteins of Eukaryote, which palyed an crucial role in maintaining the function and stability of protein. A.niger phytase was a high glycosylated protein with 9 to 11 potential glycosylation sites generally. A.niger phytases exhibited a high amino acid sequence homologies with the conserved catalytic residues, including a catalytic motif of RHGARXP and a substrate binding motif of HD. It had been found that the deglycosylated phytase could decrease the thermalstability, while the phytases expressed by different expression systems showed different glycosylation extent. Thus, glycosylation might has no direct relationship to thermalstability. Therefore, it was still unclear whether glycosylation could affect the catalytic activity and configuration stability of phytase. In order to study the effect of glycosylation to phytase characterization, a glycosylation site was changed, which provided a far-ranging notion in seeking ideal phytase with commercial interest.1. A.niger N-3 phytase gene was cloned, and 4 mutants were obtained through introducing glycosylation site (A328T and A328S),deleting glycosylation site (N326S/A328T),or changing the relative amino acids (N326D) of phytase. Then the phytases were purified, and compared with WT phytase, the mutant phytases had no obvious change. And the molecular weight were dropped from about 80 kD to 50 kD though SDS-PAGE analysis. Compared the specific activity of mutant phytases with WT, the mutant A328T increased about 14%, mutant N326D had a slight drop.2. The optimum pH study of phytase. There were two optimum pH peaks (pH 2.0 and pH 5.5) for WT recombinant phytase A.niger N-3, the activity of whichi at pH2.0 was 30% higher than at pH5.5. Although there were still two peaks, the optimum pH value shifted to 3.5 after deglycosylated by Endo Hf. It demonstrated that glycosylation could directly affect the optimum pH of phytase. Compared with WT phytase, all of the four mutants had two peaks at pH 2.5 and pH 5.5, and the relative activity had an visible increase at pH 5.5. So it could be conferred that the amino acids near HD sequence might affect the phytase's optimum pH.3. The kinetics analysis showed that the mutant A328T had a higher affinity for substrate, and the K_m value dropped by 23% compared to WT phytase. The k_cat and k_cat/K_m value increased obviously, which demonstrated that the mutant could enhance the affinity of phytase to phytate and the catalytic activity. The introduced glycosylation site might influence the glycan decoration of the two glycosylation site, which were anear to it. This may be the reason to affect the affinity of phyate.4. The thermalstability compare of the WT and mutant phytases: the phytases were treated at 85℃for 15 min, the residual activity of A328T was increased by 30%, while the mutant A328S had no change. Although both of the above two mutants were introduced a glycosylation site, the motif N-X-T was easier to be glycosylated than N-X-S motif according to Kasturi (1995). The mutants of N326D and N326S/A328T had a slight decrease in thermalstability, which was accord with the report that 326N might affect the phytase thermalstability.5. The fluorescence analysis of phytases showed that the fluorescence emission maxima had different red shift in the high temperature. There was nearly no red shift for mutant A328T at 85℃,while the mutant N326D had a about 4 nm red shift. It illuminated that mutant A328T could still keep a integrated coformation, and the mutant N326D had a tiny change on part of the phytase dimensional conformation which made the fluorescence emission group emerge more.